liquid chromatography-mass spectrometry (LC-MS)

ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-MS)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
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LC-MS (Liquid Chromatography-Mass spectrometry)
INTRODUCTION: -
Liquid chromatography (LC) is a separation process used to isolate the individual components of a
mixture. This process involves mass transfer of a sample through a polar mobile phase and non-polar
stationary phase.
Mass spectrometry (MS) ionizes atoms or molecules to facilitate their separation and detection in
accordance with their molecular masses and charges (mass to charge ratio).
LC-MS: -
The combined technique between MS and HPLC is commonly known as LC-MS. Combining the two
analytical methods reduces experimental error and improves accuracy. The application of LC-MS is
very useful in situations that involve a huge number of compounds, such as environmental effluents.
PRINCIPLE: -
✓ The Liquid Chromatography-Mass Spectrometry (LC-MS) is hyphenated analytical technique
which is combination of Liquid Chromatography (LC) and Mass Spectrometry (MS).
✓ HPLC (LC) separates the components of mixtures by passing through chromatographic
column. Generally, the separated components cannot be positively identified LC alone.
✓ Mass Spectrometry is also used for identification of unknown compounds, known compounds
and to elucidate the structure. Mass spectrometry is alone not good for identifying mixtures
because mass spectrum mixture is actually complex of overlapping spectra from separated
individual components. It is difficult to connect Liquid chromatography (LC) with Mass
spectrometry (MS). An interface is used to transfer the liquid eluents from LC to MS.LC-MS
is more significantly used in invite dissolution, bioavailability, bioequivalence and
pharmacodynamics studies.
✓ Preparative LC-MS systems can be used for rapid mass-directed purification of specific
substances from such mixtures that are important in basic research, pharmaceutical,
agrochemical, food and other industries.

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INSTRUMENTATION: -

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1. Reservoir
2. Pump and Gradient controller
3. Injector
4. Pre-column
5. Analytical Column
6. Detector

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1. Reservoir: -
• Glass or stainless-steel containers capable of holding up to 1 liter mobile phase (pure organic
solvents or aqueous solutions of salts and buffers)
• Inert to a variety of aqueous and non-aqueous mobile phases.
• Stainless steel should be avoided for use with solvents containing halide ions.
• Mobile phase is filtered to remove particulate matter and also degassed using vaccum,
sonication and sparging with helium.
2. Pump and Gradient Controller: -
a) Mechanical - 1. Reciprocating piston type- deliver at constant flow rate
Pressure variation may cause unstable baseline and therefore 2. Pulse damping device is used
b) Pneumatic- produce constant pressure, pulseless operation, pulse damping device not
required.
I) Gas displacement type- it uses direct pressure from a highly compressed gas and
force the solvent to out of a tube.
II) Amplifier type- compressed gas at lower pressure force piston to deliver
liquid
Gradient controller: - Electronic device that synchronize the operation of two pump to provide
a mobile phase mixture of desired concentration.
3. Injector: -
• It is used to introduce sample volume into the chromatographic system. Generally sample
volume from 1μL to 100μL can be injected.
• The injection volume can be increase by injector loop up to 2mL volume.
• Several injector devices are available either for manual or auto injection of the sample-
a) Septum Injector-These are used for injecting the sample through a rubber septum. This
kind of injectors cannot be commonly used, since the septum has to withstand high
pressures.
b) Stop Flow Injector- In this type the flow of mobile phase is stopped for a while & the
sample is injected through a valve.
c) Rheodyne Injector-
✓ It is the most popular injector and is widely used.
✓ This has a fixed volume of loop, for holding sample until its injected into the column,
like 20μL, 50μL or more.
✓ Through an injector the sample is introduced into the column.
✓ The injector is positioned just before the inlet of the column.
4. Precolumn: -
• To prevent the stripping of SP costed on solid material by the mobile phase and so precolumn
is coated with high percentage of stationary phase liquid to saturate the mobile phase. But
now a days banded phase chromatography is used where SP is permanently bonded to the
support material and so there is no chances of stripping.
• Trap particulate matter in mobile phase and this increase the life of main analytical column
and therefore it is called Guard column or Precolumn Filter. This is packed with SP identical
to main analytical column but particle size is greater. Length is 2 to 10 cm.
5. Analytical column: - (Heart of the LC)

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• The success or failure of analysis depends upon choice of column.
• Actual separation is takes place.
• Made up of Stainless-steel tube length is 5 to 25 cm and diameter is 2-4.6 mm.
• Column is filled with small particles 5-10 microns.
• In analytical column, solid support used is silica gel, other material which can be used are
alumina, Diatomaceous earth and activated charcoal.
• Silica gel is prepared by acidification of sodium silicate and finally washing with water
• Silanol groups are present on surface of silica gel.
• The separation is result of different components adhering to or diffusion into the packing
particles when the mobile phase is forced through column.
Normal phase chromatography -
• Stationary phase- POLAR NATURE (silica gel)
• Mobile phase- NON-POLAR
Reverse phase chromatography-
• Stationary phase- NON-POLAR NATURE
• Mobile phase- POLAR NATURE
If stationary phase is coated with polar liquid, for example: ethylene glycol, we have
to use non-polar solvent as mobile phase and chromatography will be normal phase
chromatography. If stationary phase is non polar and mobile phase is polar, it will be
Reverse Phase Chromatography (RPC).
6. Detector: -
• UV-Visible detector
• Refractive index (detects the change in turbidity)
• Fluorescence (if the analyte is fluorescent)
• Electrochemical (measures current flowing through a pair of electrodes, on which a potential
difference is imposed, due to oxidation or reduction of solute)
• Conductivity (for ions)
• Light scattering
• Mass spectrometry (HPLC-MS)

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• UV-VISIBLE DETECTOR: -
✓ Measures the ability of solutes to absorb light at a particular wavelength(s) in the ultraviolet
(UV) or visible (Vis) wavelength range.
✓ When light of a certain wavelength is directed at a flow cell, the substance inside the flow cell
absorbs the light. As a result, the intensity of the light that leaves the flow cell is less than that
of the light that enters it. An absorbance detector measures the extent to which the light
intensity decreases (i.e., the absorbance).
✓ Three Common types of UV/Vis Absorbance Detectors
➢ Fixed wavelength detectors
➢ Variable wavelength detectors
➢ Photodiode array detectors- Photo Diode Array Detector operates by simultaneously
monitoring absorbance of solutes at several different wavelengths. Light from the broad
emission source such as a deuterium lamp is collimated by an achromatic lens system so
that the total light passes through the detector cell onto a holographic grating. In this
way, the sample is subjected to light of all wavelengths generated by the lamp. The
dispersed light from the grating is allowed to fall on to a diode array. The array may
contain many hundreds of diodes and the output from each diode is regularly sampled
by a computer and stored on a hard disc.
• REFRACTIVE INDEX DETECTOR: -
Measures the overall ability of the mobile phase and its solutes to refract or bend light. Refractive
index detector measures the molecule’s ability to deflect light in a flowing mobile phase in a flow
cell relative to a static mobile phase contained in a reference cell. The amount of defection is
proportional to the concentration of the solute in the mobile phase.

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7. Ionization and Interface source: -
It is difficult to interface a liquid chromatography to a mass-spectrometry because of the necessary to
remove the solvent.
The commonly used interfaces are: -
a) Electrospray ionization (ESI)
• The LC eluent flows through a metal capillary contained within the probe.
• Droplets are formed by nebulisation of the LC flow into a spray as it leaves the electrospray
capillary.
• A charge is transferred onto the droplets by applying a large (2-5 kV) potential difference
between the electrospray capillary and counter electrode.
• The droplet size reduces by evaporating the mobile phase by the use of a heated drying gas.
• This desolvation increases charge density on the surface of the smaller droplets.
• Electric repulsion due to the charge density results in droplet fission.
• When this exceeds the surface tension of the droplet it results in coulombic fission.
• Gas-phase ions are formed as the droplet “explodes” and are sampled typically through some
form of orifice.
• The main advantage of the use of ESI for quantitative LC-MS is the formation of protonated
or de-protonated molecules with little fragmentation, ideal for selection of precursor ions and
for maximising sensitivity.
b) Atmospheric pressure chemical ionization (APCI)
In APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C) vaporizer at
atmospheric pressure. The heat vaporizes the liquid. The resulting gas-phase solvent molecules
are ionized by electrons discharged from a corona needle. The solvent ions then transfer charge
to the analyte molecules through chemical reactions (chemical ionization). The analyte ions
pass through a capillary sampling orifice into the mass analyzer. APCI is applicable to a wide
range of polar and nonpolar molecules. It rarely results in multiple charging so it is typically
used for molecules less than 1,500 u. Due to this, and because it involves high temperatures,
APCI is less well-suited than electrospray for analysis of large biomolecules that may be
thermally unstable. APCI is used with normal-phase chromatography more often than
electrospray is because the analytes are usually nonpolar.

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c) Atmospheric pressure photo ionization (APPI)
Atmospheric pressure photoionization (APPI) for LC/MS is a relatively new technique. As in
APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates photons
in a narrow range of ionization energies. The range of energies is carefully chosen to ionize as
many analyte molecules as possible while minimizing the ionization of solvent molecules. The
resulting ions pass through a capillary sampling orifice into the mass analyzer.
8. Mass analyzer: -

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Although in theory any type of mass analyzer could be used for LC/MS, four types:
• Quadrupole: -
A quadrupole mass analyzer consists of four parallel rods arranged in a square. The analyte ions
are directed down the center of the square. Voltages applied to the rods generate electromagnetic
fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a
given time. Quadrupoles tend to be the simplest and least expensive mass analyzers. Quadrupole
mass analyzers can operate in two modes:
➢ Scanning (scan) mode- the mass analyzer monitors a range of mass-to-charge ratios. In
SIM mode, the mass analyzer monitors only a few massto-charge ratios.
➢ Selected ion monitoring (SIM) mode- SIM mode is significantly more sensitive than scan
mode but provides information about fewer ions. Scan mode is typically used for qualitative
analyses or for quantitation when all analyte masses are not known in advance. SIM mode
is used for quantitation and monitoring of target compounds.
• Time-of-flight: -
In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied to all ions at
the same time, causing them to accelerate down a flight tube. Lighter ions travel faster and arrive
at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times.
Time-of flight mass analyzers have a wide mass range and can be very accurate in their mass
measurements.

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• Ion trap: -
An ion trap mass analyzer consists of a circular ring electrode plus two endcaps that together form
a chamber. Ions entering the chamber are “trapped” there by electromagnetic fields. Another field
can be applied to selectively eject ions from the trap. Ion traps have the advantage of being able
to perform multiple stages of mass spectrometry without additional mass analyzers.
• Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS): -
➢ An FT-ICR mass analyzer (also called FT-MS) is another type of trapping analyzer. Ions
entering a chamber are trapped in circular orbits by powerful electrical and magnetic fields.
When excited by a radio-frequency (RF) electrical field, the ions generate a time dependent
current. This current is converted by Fourier transform into orbital frequencies of the ions
which correspond to their mass to charge ratios.
➢ Like ion traps, FT-ICR mass analyzers can perform multiple stages of mass spectrometry
without additional mass analyzers. They also have a wide mass range and excellent mass
resolution. They are, however, the most expensive of the mass analyzers.

liquid chromatography-mass spectrometry (LC-MS)

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